Project Summary/Abstract Intratumoral genetic therapy for lung cancer We have focused investigations on augmenting systemic immune responses in non-small cell lung cancer (NSCLC) by genetic and cellular manipulation of the immune environment at the tumor site. Because lung cancer cells themselves cannot present antigen, effective professional antigen presenting cells are required. Our research group was the first to report that the intratumoral injection of dendritic cells (DC) led to systemic antitumor immune responses resulting in limitation of metastatic disease in pre-clinical lung cancer models. By altering the tumor microenvironment at the primary site, our investigations focus on restoring tumor antigen presentation and antitumor effector activities that can induce systemic antitumor immune responses and reduce metastatic disease. In 2000, we reported for the first time that C-C motif ligand-21 (CCL21, also previously referred to as secondary lymphoid tissue chemokine, exodus-2 or 6Ckine) led to potent immune- dependent antitumor responses in preclinical models of NSCLC. CCL21 is a lymphoid chemokine that is predominantly and constitutively expressed by high endothelial venules in lymph nodes and Peyer's patches and by lymphatic vessels, stromal cells in the spleen and appendix. CCL21 binds to the chemokine receptor CCR7 and is chemoattractant for mature DCs, naive and memory T cells. Our subsequent studies revealed that in vitro propagated autologous DC served as the most effective vehicle for introduction of CCL21 to the tumor site. We therefore created models in which DC were gene modified to over express the chemokine CCL21 (AdCCL21-DC) for intratumoral injection. Based on our findings in the laboratory, we have translated this work for clinical evaluation for late stage lung cancer patients. Following successful competitive peer review of this concept, the NCI Rapid Access to Investigational Drug program completed cGMP production of our adenoviral vector expressing CCL21 (Ad-CCL21). Utilizing this vector we have demonstrated that viable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved peripheral blood mononuclear cells (PBMC). This has allowed us to conduct a Recombinant Advisory Committee (RAC) and FDA-approved pilot and feasibility clinical trial in a small group of patients with inoperable NSCLC. These patients are receiving intratumoral injection of adenoviral CCL21 gene modified DC (AdCCL21-DC) and are monitored for toxicity and specific immune responses as presented in the preliminary data section. We now propose a formal phase I trial to enroll 21 patients at the VA Greater Los Angeles Healthcare Center and UCLA Ronald Reagan Medical Center in an assessment of safety, anti-tumor response and immune reactivity. In the context of this phase I trial in patients with advanced NSCLC we will determine the safety and maximum tolerated dose of intratumorally administered AdCCL21-DC. In addition, the local and systemic biological activity will be evaluated by assessment of anti-tumor immune responses, tumor immunohistochemistry, alterations in T regulatory cells and plasma cytokine profiles following intratumoral AdCCL21-DC administration. Finally, the clinical activity as reflected in reduction in tumor burden will be assessed. This is an entirely new approach to clinical immune-based intervention in lung cancer; this is the first time that humans are receiving CCL21 in any form and the first clinical trial to assess intratumoral injection of DC in lung cancer.